I. Origins of Allergic Disease Over 100 million people worldwide suffer from birch pollen allergy. However, identification of molecular determinants driving the allergic responses to Bet v 1, the major birch pollen allergen, remains elusive. In collaboration with a group in Salzburg, Austria, we examined the pollen microbiota and investigated the allergenicity of Bet v 1 upon interaction with pollen-derived compounds. At NIEHS we studied the binding of Bet v 1 to pollen derived compounds, which increased protein stability but did not induce allergic sensitization in vivo, even in the presence of mircrobial agonists. In contrast, birch pollen extracts (even those depleted of Bet v 1) strongly promoted an allergic type immune response. This finding indicates that the allergy polarizing potential of birch pollen extract is Bet v 1 independent. Sensitization to Bet v 1 is induced by an as-yet-undetermined pollen compound or mechanism in the pollen environment. These data suggest that sensitization is not exclusively linked to the intrinsic properties of individual proteins. II. Characterization of Allergens The vicilins, from peanut, walnut, cashew and pistachio are considered major allergens and are translated with leader sequences (LS) that are cleaved before yielding the mature protein. Structurally these LS were thought to be unstructured, but previous work suggested they might contain immunodominant epitopes.Linear IgE epitopes were identified using microarray data generated by printing 15-mer peptides offset by 5 amino acids on glass slides. IgE binding by peanut allergic sera was detected with a fluorescently-labeled antibody. Western blots and mass spectrometry were used to show the presence of the LS in peanut and walnut seeds. The NMR Structure of the Ara h 1 LS was determined and the IgE binding sites were modeled on this structure. The epitopes with the highest degree of IgE binding were clustered within regions that were near cysteine residues. Of the patients tested, 96% showed IgE binding to those epitopes even if they recognized no other epitopes in the Ara h 1 LS. The NMR structure showed 4 of the cysteine residues are disulfide bonded and hold together two parallel alpha helices. IgE binding is shown to be located at the junction of the c-terminal region of the alpha helices and the beginning of each flexible loop. The results indicate that cysteine residues known to confer high structural stability to allergens may also coincide with areas of increased IgE binding frequency and intensity in Ara h 1 LS. The leader sequence contain multiple immunodominant epitopes and may be important in cross-reactivity and nut allergy. III. Adaptive Immune response The defining characteristic of allergy is the generation of IgE antibodies, which leads to patient symptoms. We wish to probe more fundamental properties of the antibody response. It is suggested that by better understanding and characterizing the antibodies of all types new treatment modalities, or safer therapies can be developed. Previously, we determined the first structure of the major peanut allergen Ara h 2. Ara h 2 is recognized by more than 90% of peanut allergic patients and sensitivity to Ara h 2 is measurable risk factor for peanut induced anaphylaxis. We have initiated a collaboration with researchers at Harvard University who have been studying the antibody production of peanut allergic patients in response to oral immunotherapy. By isolating B-cells from the patients and sequencing immunoglobulin genes, they found evidence that separate patients were honing in on similar regions of Ara h 2. We have received 5 of these antibody clones and are working to determine structures of antibody fragments in complex with Ara h 2. So far, we tested the production of all 5, and have scaled up production. The epitope information derived from these complexes will be useful in understanding the targets of the adaptive immune response of patients during peanut immunotherapy. Directly examining human IgE in complex an allergen is technically challenging for a number of reasons. First of all, the memory cells that make IgE are extremely rare in sera. Scott Smith at Vanderbilt University has recently developed a technique to clone these rare cells and produce full length human IgE. We have acquired 4 human monoclonal IgE antibodies against the major dust mite allergen Der p 2 to study as a collaborative project. In the past year we have been measuring the interactions of the human IgE with Der p 2 via NMR technologies. We hope that this pioneering technique could be generalized to study the response to other allergens, and may be applicable to other antibody types besides IgE. The results will help us better understand the human immune response to allergens like Der p 2 with a hope using the information to design hypo-allergens that will improve allergy immunotherapy, better known as allergy-shots.